A correct assessment of cancer risks associated with human exposure to chemicals is of great importance from both public health and economic perspectives. In this regard, accurate and reliable tests that estimate the carcinogenic potency of compounds are indispensable. The two-year rodent carcinogenicity bioassay is the gold standard for carcinogenic screenings of newly developed drugs and other chemical compounds, but high costs, long experimental periods and European (REACH) and U.S. (ICCVAM Authorization Act) policies promote the search for alternative assays (1-5). In addition, because of raising ethical concerns regarding animal use in scientific procedures, organizations like EURL ECVAM (European Union Reference Laboratory for the Validation of Alternative Methods) and ICCVAM (Interagency Coordinating Committee on the Validation of Alternative Methods) have been founded to validate alternative approaches in accordance with the 3Rs (Replace, Reduce and Refine) Declaration, which includes the use of organisms with limited sentience such as invertebrates.
A challenge for the development of alternative test methods is that, besides an adequate estimation of carcinogenicity, compounds need to be classified according to their presumed predominant mechanism of action into genotoxic and non-genotoxic carcinogens, which entail different assessments of human cancer risk. Genotoxic carcinogens disturb the genomic integrity directly by interacting with the DNA and/or the cellular apparatus, and are assumed to have a low-dose linearity without dose thresholds in their carcinogenic effects. Non-genotoxic carcinogens alter gene expression indirectly and promote tumor growth by interfering with a variety of cellular processes associated with suppressed apoptosis and increased cell proliferation (6-8). A major difficulty in distinguishing these classes of carcinogens is the comparison of exposure conditions, since multiple signaling pathways are deregulated during non-genotoxic carcinogenesis, whereby carcinogenic effects only occur if a certain threshold dose is reached (7-10). The understanding and prediction of non-genotoxic carcinogens is substantially complicated by their compound-specific mechanisms of action (7, 10).
The present alternative, in vitro and short term in vivo assays, identify the majority of genotoxic carcinogens, although improvements in the predictive capacity are still needed to diminish false positive and negative results. Standard in vitro assays for the detection of genotoxic compounds produce up to 70% and more of irrelevant positive results, which require expensive and time-consuming follow-up in vitro and in vivo testing. Another drawback of these tests is the sensitivity and reliability in detecting non-genotoxic carcinogens, which represent up to 25% of Class I human carcinogens according to the International Agency for Research on Cancer (IARC) (3, 5, 7, 11-13). A better understanding of cellular and molecular events involved in non-genotoxic carcinogenesis is needed and, given the extensiveness and complexity of these processes, in vitro detection methods may not be sophisticated enough to cover the full carcinogenicity response.
Flatworms are promising organisms for predictive carcinogenicity/genotoxicity screenings for the following reasons: (1) Their remarkable regeneration capacity, rebuilding missing body parts within about one week, enables the study of carcinogen-induced responses during the regeneration of multiple tissues (14, 15). Since massive cell proliferation is a prerequisite for both regeneration and carcinogenesis, the organism's cellular response to carcinogen exposures is accelerated during the process of regeneration (16-18). (2) Their experimentally accessible stem cells enable in vivo studies of carcinogen-induced responses of pluripotent, adult stem cells within the entire animal. (3) The main characteristics of chemically induced carcinogenesis, namely, the initiation and promotion stage of neoplastic formation, are described in flatworms and several underlying cancer-related genes and signaling pathways have been identified, e.g., PTEN, FOXO, caspases, cyclins, (PI3K)-Akt pathway, RAS pathway, p53 pathway, and MAPK pathway (19-24).
Stalmans et al. (25) further disclose an in vivo flatworm carcinogenicity bioassay, in which stem cell proliferation is used as an endpoint to assess the carcinogenic potential of compounds. However, it is completely unknown whether flatworms can be used to detect whether a compound is a non-genotoxic carcinogen and/or whether flatworms can be used to distinguish non-genotoxic carcinogens from genotoxic carcinogens.